It has become evident that innate immunity plays an active role in maintenance of the health of the middle ear, and is also actively engaged during otitis media (OM). Antimicrobial peptides (APs) are key components of the innate immune system, providing first-line inactivation of microbes on the vast epithelial surfaces that line the respiratory, gastrointestinal and urogenital tracts. The potent antimicrobial activity of these cationic polypeptides, can be additive and is often synergistic, providing the highly effective mechanism of action of the innate immune system. It is now clear however, that APs are not simply antimicrobial, and in fact, microbial killing may not even be their most primary function, rather there is increasing evidence of the pivotal role of APs in regulation of the acquired immune response. Recently, it has also begun to be appreciated that the commensal microorganisms living on mucosal surfaces stimulate epithelial cells to produce APs, and thus contribute to host maintenance of defensive mucosal barriers and homeostasis. This latter point is particularly relevant to our renewal application, because OM is not caused by highly virulent microorganisms; OM is instead caused by a subset of the commensal bacteria that comprise the normal flora of the pediatric naso- pharynx (NP). However, when host airway defenses are compromised, most typically by upper respiratory tract (URT) viruses, these bacteria can behave as opportunistic pathogens and gain access to the now poorly defended middle ear, a situation that closely parallels that of the gut, wherein the molecular crosstalk that occurs between the normal microbiota and the epithelial cells that line the gastrointestinal tract has been well- studied. Understanding how host and microbe have co-evolved to exist in harmony during colonization (and in fact, how these microbes have likely helped to shape the innate immune system of the uppermost airway and vice versa) is essential to our then being able to understand what goes `wrong' early in the disease course of OM, as well as what allows return to homeostasis and health. It is thereby important to continue to study the innate immune system of the uppermost airway, which includes the NP, Eustachian tube and middle ear. Toward this goal, we propose to investigate the role of innate immunity in: 1) health (i.e. during colonization) at the level of the molecular crosstalk that occurs between nontypeable Haemophilus influenzae (NTHI) and the mucosal epithelium of the NP; 2) the polymicrobial disease OM (i.e. `normal' OM), with a particular focus on how dysregulated expression of effectors of innate immunity and altered dendritic cell function(s) induced by the URT viruses contributes to the disease process; 3) the OM disease extremes - proneness and chronicity, with a particular focus on how copy number variations in genes that encode alpha- and beta-defensin family members contribute to proneness. We will also study how APs might first inadvertently contribute to chronicity by inducing NTHI to form a biofilm, but later participate in its resolution and 4 to utilize our enhanced under- standing of innate immunity in the uppermost airway to develop novel methods to treat and/or prevent OM. PUBLIC HEALTH RELEVANCE: Due to the continuing need for improved methods to treat and/or prevent otitis media (OM), the desire for less invasive, more targeted pediatric vaccines and the realization that our understanding of both the microbiology and immunology of acute and chronic OM is incomplete, it is imperative that we seek a greater understanding of how the body defends the uppermost airway, and that methods to potentially augment these defense mechanisms be identified and developed further as a novel approach for the treatment and/or prevention of OM. Gaining this improved understanding will potentially allow us to develop therapeutic approaches that are locally restricted and site-specific, as well as facilitate the refinement of vaccine formulations so as to enhance their efficacy. These approaches could add tremendously to our available arsenal for fighting acute and chronic OM and importantly, could potentially provide a mechanism to intervene at a very early state in disease process- colonization of the NP, thereby significantly reducing the morbidity associated with this highly prevalent pediatric disease.